The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further elongation and desaturation of linoleic acid (LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg et al., Amer. J. Clin. Nutr. 28:958-966 (1975); Dyerberg et al., Lancet. 2(8081):117-119 (1978); Shimokawa, H., World Rev. Nutr. Diet 88:100-108 (2001); von Schacky et al., World Rev. Nutr. Diet 88:90-99 (2001)). Numerous other studies document wide-ranging health benefits conferred by administration of omega-3 and/or omega-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
Today, a variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production via numerous divergent efforts. Although the natural PUFA-producing abilities of the host organisms are sometimes essential to a given methodology, genetic engineering has also proven that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs. Whether this effect is the result of natural abilities or recombinant technology, production of arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) all require expression of either the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid (EDA; 20:2 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3)) or the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of γ-linoleic acid (GLA; 18:3 ω-6) and/or stearidonic acid (STA; 18:4 ω-3) (FIG. 1). A delta-6 elongase is also known as a C18/20 elongase.
The delta-8 desaturase enzymes identified thus far have the ability to convert both EDA to dihomo-γ-linolenic acid (DGLA; 20:3) and ETrA to eicosatetraenoic acid (ETA; 20:4) (wherein ARA are EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a delta-5 desaturase, while DHA synthesis requires subsequent expression of an additional C20/22 elongase and a delta-4 desaturase).
Based on the role delta-8 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has been effort to identify and characterize these enzymes. Initial efforts on the isolation and characterization of delta-8 desaturases from Euglena gracilis; and, several sequence variations within the Euglena gracilis delta-8 desaturase have been reported (see, e.g., Wallis et al., Arch. Biochem. and Biophys. 365(2):307-316 (1999); PCT Publication No. WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO 2004/057001). Also, Applicants' Assignee's co-pending applications having U.S. application Ser. Nos. 11/166,003 and 11/166,993 filed Jun. 24, 2005, respectively (PCT Publication Nos. WO 2006/012325 and WO 2006/012326; both published Feb. 2, 2006)) discloses amino acid and nucleic acid sequences for a Euglena gracilis delta-8 desaturase.
More recently, PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885). Sayanova et al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 delta-8 desaturase. Also, Applicants' Assignee's co-pending application having U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S. patent application Ser. No. 11/876,115 (filed Oct. 22, 2007; discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf—gymnastica CCMP1594.
Based on the utility of expressing delta-8 desaturases in conjunction with delta-9 elongases, there has also been effort to identify and characterize delta-9 elongases from various sources. Most delta-9 elongase enzymes identified so far have the ability to convert both LA to EDA and ALA to ETrA (wherein DGLA and ETA are subsequently synthesized from EDA and ETrA, respectively, following reaction with a Δ8 desaturase; ARA and EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a Δ5 desaturase; and, DHA synthesis requires subsequent expression of an additional C20/22 elongase and a Δ4 desaturase). A delta-9 elongase from Isochrysis galbana has been publicly available (described in GenBank Accession No. AAL37626, as well as PCT Publication No. WO 02/077213). Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; discloses a delta-9 elongase from Euglena gracilis. Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/601,564 filed Nov. 16, 2006, discloses a delta-9 elongase from Eutreptiella sp. CCMP389.
Most delta-5 desaturase enzymes identified so far have the primary ability to convert dihomo-gamma-linolenic acid [20:3, DGLA] to ARA, with secondary activity in converting eicosatetraenoic acid [20:4, ETA] to EPA (where DHA is subsequently synthesized from EPA following reaction with an additional C20/22 elongase and a delta-4 desaturase). The delta-5 desaturase has a role in both the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of gamma-linoleic acid [“GLA”; 18:3 ω-6] and/or stearidonic acid [“STA”; 18:4 ω-3]) and the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid [“EDA”; 20:2 ω-6] and/or eicosatrienoic acid [“ETrA”; 20:3 ω-3]) (FIG. 1).
Furthermore, based on the role delta-5 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has also been an effort to identify and characterize these enzymes from various sources. As such, delta-5 desaturases have been disclosed in both the open literature (e.g., GenBank Accession Nos. AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654 and AB022097) and the patent literature (e.g., U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183).
Applicants' Assignee has a number of patent applications concerning the production of PUFAs in oleaginous yeasts (i.e., Yarrowia lipolytica), including: PCT Publication Nos. WO 2004/101757 and WO 2004/101753 (both published Nov. 25, 2004); U.S. application Ser. No. 11/265,761 (filed Nov. 2, 2005); U.S. application Ser. No. 11/264,784 (filed Nov. 1, 2005); and U.S. application Ser. No. 11/264,737 (filed Nov. 1, 2005).
Relatedly, PCT Publication No. WO 2004/071467 (published Aug. 26, 2004) concerns the production of PUFAs in plants, while PCT Publication No. WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants; both are Applicants' Assignee's copending applications.